Crystalline form of tegavivint, method of preparation, and use thereof

ABSTRACT

The present invention relates to crystalline forms of (9E,10E)-2,7-bis((3,5-dimethylpiperidin-1-yl)sulfonyl)anthracene-9,10-dione dioxime, pharmaceutical compositions comprising the crystalline form, processes for preparing the crystalline form and methods of use therefore.

FIELD OF THE INVENTION

The present invention relates to crystalline forms of tegavivint, aka,(9E,10E)-2,7-bis((3,5-dimethylpiperidin-1-yl)sulfonyl)anthracene-9,10-dionedioxime, pharmaceutical compositions comprising the crystalline form,processes for preparing the crystalline form, and methods of usethereof.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States. Itpresents complex challenges for the development of new therapies. Canceris characterized by the abnormal growth of malignant cells that haveundergone a series of genetic changes that lead to growth of tumor massand metastatic properties.

Beta-catenin (β-catenin) is part of a complex of proteins thatconstitute adherens junctions (AJs). AJs are necessary for the creationand maintenance of epithelial cell layers by regulating cell growth andadhesion between cells. β-catenin also anchors the actin cytoskeletonand may be responsible for transmitting the contact inhibition signalthat causes cells to stop dividing once the epithelial sheet iscomplete.

Wnt/β-catenin pathway has been shown to play a role in cancer. Aberrantβ-catenin signaling plays an important role in tumorigenesis. Inparticular, colorectal cancer is estimated to have greater than 80%mutations in the β-catenin pathway, leading to unregulated oncogenicsignaling. Aberrant β-catenin signaling has been shown to be involved invarious cancer types, including but not limited to, melanoma, breast,lung, colon, liver, gastric, myeloma, multiple myeloma, chronicmyelogenous leukemia, chronic lymphocytic leukemia, T-cell non-Hodgkinlymphomas, colorectal and acute myeloid leukemia (AML) cancers. Further,aberrant Wnt/β-catenin signaling has been found in a large number ofother disorders, including osteoporosis, osteoarthritis, polycystickidney disease, diabetes, schizophrenia, vascular disease, cardiacdisease, hyperproliferative disorders, neurodegenerative diseases, andfibrotic diseases including but not limited to idiopathic pulmonaryfibrosis (IPF), Dupuytren's contracture, Nonalcoholic steatohepatitis(NASH), and others. Myeloproliferative neoplasms (MPNs) are a closelyrelated group of hematological malignancies in which the bone marrowcells that produce the body's blood cells develop and functionabnormally. The three main myeloproliferative neoplasms are PolycythemiaVera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis(PMF). A gene mutation in JAK2 is present in most PV patients and 50% ofET and PMF patients. The beta catenin pathway is activated in MPN inmany cases and required for survival of these cells.

Tegavivint and related compounds are described, for example, in U.S.Pat. No. 8,129,519. Tegavivint has the following structural formula:

The chemical name is(9E,10E)-2,7-bis((3,5-dimethylpiperidin-1-yl)sulfonyl)anthracene-9,10-dionedioxime

The molecular formula of tegavivint is C₂₈H₃₆N₄O₆S₂

The molecular mass of tegavivint is 588.20763 amu.

The small scale chemical synthesis of tegavivint had been disclosed inU.S. Pat. No. 8,129,519. The drug substance/Active PharmaceuticalIngredient (API) has good chemical and physical stability. However,there is a major concern about physical stability of nanoparticleformulations of tegavivint over time that can manifest as crystal growth(Oswald ripening), or a polymorphic change, which can result in theincrease in large particle count, or in generation of unfavorableparticle morphology during long term storage of the formulated drug.Thus, there remains a need to perform crystal investigation to exploresuitable/relevant polymorph(s) of tegavivint that would be feasible formilling and formulation development to yield a formulation with goodlong term physical stability. The present invention advantageouslyaddresses this need.

SUMMARY OF THE INVENTION

The present application discloses an invention to address the foregoingchallenges and need by providing a crystalline single polymorphic formof tegavivint, referred to throughout this application as Form IV. Thecurrent formulation of tegavivint is a nanosuspension created utilizinga milling process. While Form I (BC-2059 obtained from chemicalsynthesis as is) has been currently utilized as the starting materialfor the milling process and the end product obtained from milling isnanosuspension of Form I. However, the inventors of present inventionhave unexpectedly found that there are specific advantages of utilizingForm IV (in comparison to Form I) as the starting material for themilling process to prepare a nanosuspension of tegavivint.

The main advantage is that Form IV is sufficiently unstable so that FormIV gets converted to Form I when milled at an elevated temperature (60°C.). Thus, the system will undergo a full solvent-mediatedrecrystallization from Form IV to Form I. The crystals for Form I willgrow “bottom-up” as they are milled, so the chance of getting anyunmilled larger crystals would be significantly diminished. In otherwords, it is beneficial to utilize Form IV as the starting materialbecause it will eventually be converted to Form I and the only Form Icrystals would come from re-crystallization from Form IV. Thus,facilitating suspension with a single polymorph form generated throughmilling at elevated temperatures would in turn enhance the stability ofthe suspension.

Thus, in one embodiment, the invention provides a crystalline form oftegavivint, referred to as Form IV which has an X-ray powder diffractionpattern (XRPD) comprising diffraction peaks having ° 2θ angles selectedfrom the group consisting of 5.0+−0.2°; 7.5+−0.2°; 7.7+−0.2°;10.2+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 18.0+−0.2°; 20.0+−0.2°;20.5+−0.2°; and 22.2+−0.2°.

In one embodiment, Form IV can be a single crystal.

In one embodiment, Form IV is a trihydrate.

In another embodiment, Form IV has an endothermic peak at about 115.9°C.

In another embodiment, Form IV has an onset of exothermic peak at about147.1° C.

In yet another embodiment, exothermic decomposition of Form IV starts atabout 280° C.

In yet another embodiment, the invention provides a nanosuspension oftegavivint wherein the nanosuspension was prepared by a processcomprising using Form IV as the starting material and milling Form IV ata temperature of between about 40° C. and about 60° C., most preferablyat about 60° C.

In one embodiment, if the milling process is done at temperature of lessthan about 60° C., the nanosuspension has to further undergo theannealing process at or above 60° C.

In another embodiment of the invention, pharmaceutical compositions areprovided for use in the methods comprising stable nanosuspensions ofForm I prepared using Form IV as the starting material, andpharmaceutically acceptable excipient.

In another embodiment of the invention, provided herein are methods forpreventing, treating or ameliorating cancer or tumor metastasis in amammal in need thereof comprising administering to said mammal aneffective amount of the compositions of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an X-ray powder diffraction (XRPD) pattern of Form Iof tegavivint.

FIG. 2 illustrates a differential scanning calorimetry (DSC) curve andThermogravimetric Analysis (TGA) curve of Form I.

FIG. 3 shows a Polarized Light Microscopy (PLM) image of Form I.

FIG. 4 shows XRPD patterns overlay of Form II preparation.

FIG. 5 shows XRPD patterns overlay of Form III preparation.

FIG. 6 illustrates a DSC curve and TGA curve of Form III.

FIG. 7 shows a PLM image of Form III.

FIG. 8 shows XRPD patterns overlay of Form III samples after drying.

FIG. 9A shows XRPD patterns of Form IV samples.

FIG. 9B shows a DSC curve and TGA curve of Form IV.

FIG. 9C shows a Dynamic Vapor Sorption (DVS) profile of Form IV.

FIG. 9D shows a PLM image of Form IV.

FIG. 9E shows Variable Temperature X-ray Powder Diffraction (VT-XRPD)profile of Form IV.

FIG. 9F shows XRPD patterns overlay of Form IV sample after VT-XRPD andexposure to ambient conditions.

FIG. 10 shows XRPD pattern of Form V.

FIG. 11 shows XRPD pattern of Form VI.

FIG. 12A illustrates XRPD pattern of amorphous sample.

FIG. 12B shows a modulate DSC (mDSC) curve of amorphous sample.

FIG. 13A shows XRPD pattern of solid obtained from slurry competition inwater.

FIG. 13B shows XRPD pattern of solid obtained from slurry competition inACN/water (1:1, v/v).

FIG. 13C shows XRPD pattern of solid obtained from slurry competition inACN/water (1:3, v/v).

FIG. 14A is a Particle Size Distribution (PSD) plot of Form I samplemilled at 5° C.

FIG. 14B is a PSD plot of Form I sample milled at RT.

FIG. 14C is a PSD plot of Form I sample milled at 60° C.

FIG. 14D is a PSD plot of Form IV sample milled at 5° C.

FIG. 14E is a PSD plot of Form IV sample milled at RT.

FIG. 14F is a PSD plot of Form IV sample milled at 60° C.

FIG. 14G is a PLM image of Form I and Form IV.

FIG. 14H is a PLM image of Form I sample milled at 5° C.

FIG. 14I is a PLM image of Form I sample milled at RT.

FIG. 14J is a PLM image of Form I sample milled at 60° C.

FIG. 14K is a PLM image of Form IV sample milled at 5° C.

FIG. 14L is a PLM image of Form IV sample milled at RT.

FIG. 14M is a PLM image of Form IV sample milled at 60° C.

FIG. 14N shows XRPD patterns overlay of Form I sample milled at 5° C.

FIG. 14O shows XRPD patterns overlay of Form I sample milled at RT.

FIG. 14P shows XRPD patterns overlay of Form I sample milled at 60° C.

FIG. 14R shows XRPD patterns overlay of Form IV sample milled at 5° C.

FIG. 14S shows XRPD patterns overlay of Form IV sample milled at RT.

FIG. 14T shows XRPD patterns overlay of Form IV sample milled at 60° C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to crystalline forms of tegavivint. Inparticular, the present invention relates to a crystalline formdesignated Form IV of tegavivint, pharmaceutical compositions comprisingthe crystalline form, processes for preparing the crystalline form andmethods of use thereof.

In one embodiment, the crystalline form of tegavivint is designated asForm IV, which has an X-ray powder diffraction pattern (XRPD) comprisingdiffraction peaks having ° 2θ angle values independently selected fromthe group consisting of 5.0+−0.2°; 7.5+−0.2°; 14.8+−0.2°; 15.2+−0.2°;15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°.

In one embodiment, Form IV has an XRPD comprising diffraction peakshaving ° 2θ angle values independently selected from the groupconsisting of 5.0+−0.2°; 7.5+−0.2°; 7.7+−0.2°; 14.8+−0.2°; 15.2+−0.2°;15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°.

In one embodiment, Form IV has an XRPD comprising diffraction peakshaving ° 2θ angle values independently selected from the groupconsisting of 5.0+−0.2°; 7.5+−0.2°; 7.7+−0.2°; 10.2+−0.2°; 14.8+−0.2°;15.2+−0.2°; 15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°.

In another embodiment, Form IV has an XRPD comprising diffraction peakshaving ° 2θ angle values independently selected from the groupconsisting of 5.0+−0.2°; 7.5+−0.2°; 7.7+−0.2°; 10.2+−0.2°; 14.8+−0.2°;15.2+−0.2°; 15.4+−0.2°; 18.0+−0.2°; 20.0+−0.2°; and 22.2+−0.2°.

In another embodiment, Form IV has an XRPD comprising diffraction peakshaving ° 2θ angle values independently selected from the groupconsisting of 5.0+−0.2°; 7.5+−0.2°; 7.7+−0.2°; 10.2+−0.2°; 14.8+−0.2°;15.2+−0.2°; 15.4+−0.2°; 18.0+−0.2°; 20.0+−0.2°; 20.5+−0.2°; and22.2+−0.2°.

In another embodiment, Form IV has an XRPD pattern substantially asshown in FIG. 9A.

In another embodiment, Form IV is characterized by having an endothermwith a peak maximum at approximately 115.9° C. by differential scanningcalorimetry (DSC).

In another embodiment, Form IV is characterized by having an onset ofexothermic peak at approximately 147.1° C. by DSC.

In one embodiment, Form IV is characterized by having a DSC thermogramsubstantially as shown in FIG. 9B.

In one embodiment, Form IV is a trihydrate which is isolated fromsolvents with high water activity. The crystal morphology is needles.The trihydrate dehydrates thermally with the half-point of dehydrationat about 60° C.

In yet another embodiment, the invention provides a nanosuspension oftegavivint wherein the nanosuspension was prepared by a processcomprising using Form IV as the starting material and milling Form IV ata temperature of between about 40° C. and about 60° C., most preferablyat about 60° C.

In one embodiment, if the milling process is done a temperature of lessthan about 60° C., the nanosuspension has to further undergo theannealing process at or above 60° C.

In another embodiment of the invention, pharmaceutical compositions areprovided for use in the methods comprising stable nanosuspensions ofForm I prepared using Form IV as the starting material, andpharmaceutically acceptable excipient.

In another embodiment of the invention, provided herein are methods forpreventing, treating or ameliorating cancer or tumor metastasis in amammal in need thereof comprising administering to said mammal aneffective amount of the compositions of the invention.

The crystalline forms of tegavivint may be formulated by any method wellknown in the art and may be prepared for administration by any route,including, without limitation, parenteral, oral, sublingual,transdermal, topical, intranasal, intratracheal, or intrarectal. Incertain embodiments, the crystalline form of tegavivint is administeredintravenously in a hospital setting. In one embodiment, administrationmay be by the oral route.

The characteristics of the carrier will depend on the route ofadministration. As used herein, the term “pharmaceutically acceptable”means a non-toxic material that is compatible with a biological systemsuch as a cell, cell culture, tissue, or organism, and that does notinterfere with the effectiveness of the biological activity of theactive ingredient(s). Thus, compositions may contain, in addition to theinhibitor, diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The preparation ofpharmaceutically acceptable formulations is described in, e.g.,Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, MackPublishing Co., Easton, Pa., 1990.

The pharmaceutical compositions comprising a crystalline form oftegavivint may be used in the methods of use described herein.

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount without causing serious toxic effectsin the patient treated. The effective dosage range of thepharmaceutically acceptable derivatives can be calculated based on theweight of the parent compound to be delivered. If the derivativeexhibits activity in itself, the effective dosage can be estimated asabove using the weight of the derivative, or by other means known tothose skilled in the art.

In some embodiments of any of the methods described herein, beforetreatment with the compositions or methods of the invention, the patientmay be treated with one or more of a chemotherapy, a targeted anticanceragent, radiation therapy, and surgery, and optionally, the priortreatment was unsuccessful; and/or the patient has been administeredsurgery and optionally, the surgery was unsuccessful; and/or the patienthas been treated with a platinum-based chemotherapeutic agent, andoptionally, the patient has been previously determined to benon-responsive to treatment with the platinum-based chemotherapeuticagent; and/or the patient has been treated with a kinase inhibitor, andoptionally, the prior treatment with the kinase inhibitor wasunsuccessful; and/or the patient was treated with one or more othertherapeutic agent(s).

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents, patent applications,and publications referred to herein are incorporated by reference.

As used herein, “tegavivint” refers to(9E,10E)-2,7-bis((3,5-dimethylpiperidin-1-yl)sulfonyl)anthracene-9,10-dionedioxime.

As used herein, the term “Form IV” or “Crystalline Form IV” when usedalone refers to Crystalline Form IV of (9E,10E)-2,7-bis((3,5-dimethylpiperidin-1-yl)sulfonyl)anthracene-9,10-dione dioxime.

As used herein, the term “subject,” “individual,” or “patient,” usedinterchangeably, refers to any animal, including mammals such as mice,rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses,primates, and humans. In some embodiments, the patient is a human. Insome embodiments, the subject has experienced and/or exhibited at leastone symptom of the disease or disorder to be treated and/or prevented.In some embodiments, the subject is suspected of having a multi-tyrosinekinase-associated cancer.

As used herein, a “therapeutically effective amount” of a crystallineform of tegavivint is an amount that is sufficient to ameliorate, or insome manner reduce a symptom or stop or reverse progression of acondition, or negatively modulate or inhibit the activity of amutli-tyrosine kinase. Such amount may be administered as a singledosage or may be administered according to a regimen, whereby it iseffective.

As used herein, “treatment” means any manner in which the symptoms orpathology of a condition, disorder or disease are ameliorated orotherwise beneficially altered. Treatment also encompasses anypharmaceutical use of the compositions herein.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular pharmaceutical composition refers to anylessening, whether permanent or temporary, lasting or transient that canbe attributed to or associated with administration of the composition.

As used herein, the term “about” when used to modify a numericallydefined parameter (e.g., the dose of a crystalline form of tegavivintdetailed herein or a pharmaceutically acceptable salt thereof, or thelength of treatment time described herein) means that the parameter mayvary by as much as 10% below or above the stated numerical value forthat parameter. For example, a dose of about 5 mg/kg may vary between4.5 mg/kg and 5.5 mg/kg. “About” when used at the beginning of a listingof parameters is meant to modify each parameter. For example, about 0.5mg, 0.75 mg or 1.0 mg means about 0.5 mg, about 0.75 mg or about 1.0 mg.Likewise, about 5% or more, 10% or more, 15% or more, 20% or more, and25% or more means about 5% or more, about 10% or more, about 15% ormore, about 20% or more, and about 25% or more.

As used herein, the term “about” when used in reference to XRPD peakpositions refers to the inherent variability of peaks depending on thecalibration of the instrument, processes used to prepare the crystallineforms of the present invention, age of the crystalline forms and thetype of instrument used in the analysis. The variability of theinstrumentation used for XRPD analysis was about ±0.2° 2θ.

As used herein, the term “about” when used in reference to DSCendothermic peak onset refers to the inherent variability of peaksdepending on the calibration of the instrument, method used to preparethe samples of the present invention, and the type of instrument used inthe analysis. The variability of the instrumentation used for DSCanalysis was about ±2° C.

General Methods

The general methods outlined below were used in the exemplifiedExamples, unless otherwise noted.

Crystalline forms of the present invention may be prepared using avariety of methods well known to those skilled in the art includingcrystallization or recrystallization from a suitable solvent or bysublimation. A wide variety of techniques may be employed, includingthose in the exemplified Examples, for crystallization orrecrystallization including evaporation of a water-miscible or awater-immiscible solvent, crystal seeding in a supersaturated solventmixture, decreasing the temperature of the solvent mixture, or freezedrying the solvent mixture.

In the present invention, crystallization may be done with or withoutcrystal seed. The crystal seed may come from any previous batch of thedesired crystalline form. The addition of crystal seed may not affectthe preparation of the crystalline forms in the present invention.

The sample was recovered after completion of the isotherm andre-analyzed by XRPD.

Abbreviations and Acronyms

Category Abbreviations Full Name/Description Analytical DSC DifferentialScanning Calorimetry Techniques DVS Dynamic Vapor Sorption NMR NuclearMagnetic Resonance PLM Polarized light microscopy PSD Particle SizeDistribution TGA Thermogravimetric Analysis XRPD X-ray PowderDiffraction VT-XRPD Variable Temperature X-ray Powder DiffractionSolvent ACN Acetonitrile CHCl₃ Chloroform DMF Dimethylforamide DMSODimethylsulfoxide EtOAc Ethyl acetate EtOH Ethanol IPA Isopropyl alcoholMEK Methyl ethyl ketone MeOH Methanol MTBE Methyl-tert-butyl ether THFTetrahydrofuran Other RT Room temperature v/v percent volume ratio

The following Examples are intended to illustrate further certainembodiments of the invention and are not intended to limit the scope ofthe invention.

Example 1 Investigation of Form I of Tegavivint

This Example illustrates the investigation of Form I of tegavivint.

Starting material (Form I of tegavivint) was characterized by XRPD, TGA,DSC and PLM. XRPD pattern displayed in FIG. 1 showed the startingmaterial was crystalline and confirmed to be Form I. TGA and DSC curvesare displayed in FIG. 2. A weight loss of 0.4% up to 150° C. wasobserved on TGA curve, and DSC result showed no melting endotherm beforedecomposition. Based on the characterization results, Form I wasspeculated to be an anhydrate. PLM image displayed in FIG. 3 showedirregular fine particles with partial aggregation for Form I sample.

Example 2 Preparation of Form II of Tegavivint

Attempts to prepare Form II of tegavivint were performed in fourconditions. The detailed results are shown in FIG. 4 and Table 1. Theresults suggested that Form II was quite challenging to be re-preparedor metastable.

TABLE 1 Summary of Form II Preparation Attempts Anti-solvent, ID MethodSolvent, mL mL Results 803759-05-A1 Anti-solvent EtOH, 2.5 H₂O, 2 FormIII addition 803759-05-A2 Anti-solvent EtOH, 2.5 H₂O, 2 Form I addition803759-05-A3 Solution vapor EtOH, 2 H₂O Form I diffusion 803759-05-A4Slow EtOH, 2 n-heptane, 2 Form I evaporation

Example 3 Preparation of Form III of Tegavivint

Form III samples (803759-03-A and 803759-05-A1) were prepared viaanti-solvent addition in MeOH/H₂O and EtOH/H₂O systems, and the XRPDresults are displayed in FIG. 5. The TGA/DSC results of Form III(803759-05-A1) are displayed in FIG. 6. A weight loss of 8.2% up to 100°C. was observed on TGA. One endotherm at 64.5° C. and one exotherm at158.6° C. were observed before decomposition on DSC. The PLM imagedisplayed in FIG. 7 showed needle-like and fine particles withaggregation for Form III sample (803759-05-A1).

Since Form III samples could be obtained from different solvent systemsand converted to Form IV after vacuum drying at RT or Form V afterexposure to ambient condition at RT (FIG. 8), Form III might beisomorphic.

Example 4 Preparation of Form IV of Tegavivint

Form IV (803759-13-B) sample was re-prepared at 2-g scale. The detailedprocedures were as follows:

1. Weigh 2.0 g 803759-01-A sample into a 1-L reactor.

2. Charge 200 mL EtOH, and stir at RT with 300 rpm to obtain a clearsolution

3. Charge 100 mL water.

4. Add 90.2 mg Form IV seed, a suspension was observed.

5. Charge 100 mL water over 1 h.

6. Keep slurry for 2 h.

7. Filter and test XRPD of the wet cake.

8. Transfer the wet cake into a 1-L reactor, charge 200 mL water andslurry overnight.

9. Filter and vacuum dry for 4 hrs. 1.9 g solids were obtained(803759-13-B, Form IV).

The XRPD results of Form IV are displayed in FIG. 9A. The TGA/DSCresults were displayed in FIG. 9B. A weight loss of 8.4% up to 150° C.could be observed on TGA. One endotherm at 115.9° C. (peak) and oneexotherm at 147.1° C. (onset) before decomposition were observed on DSC.DVS result displayed in FIG. 9C showed that: 1) two platforms wereobserved indicating two potential hydrate forms existing. 2) A wateruptake of 9.1% was observed at 25° C./80% RH, which was consistent withTGA weight loss of Form IV. The PLM image displayed in FIG. 9D showedneedle-like particles was observed for Form IV sample (803759-13-B).

VT-XRPD test was employed for further investigation of Form IV, theresults displayed in FIG. 9E and FIG. 9F showed that 1) Form IV samplepartially converted to a new form at 30° C. under N2, and the new formwas named as Form VI. 2) After heating to 75 and 120° C., pure Form VIwas observed. 3) After cooling to 30° C., Form VI was still observed. 4)After exposure to ambient condition, Form VI converted to Form IVquickly. Thus, Form IV is believed to be a hydrate.

Example 5 Preparation of Form V of Tegavivint

Form V sample (803759-03-A 22Apr) was obtained via drying Form IIIsample (803759-03-A) at ambient condition, the XRPD pattern wasdisplayed in FIG. 10. The TGA and DSC data were not collected due tolimited solid.

Example 6 Preparation of Form VI of Tegavivint

Form VI sample (803759-02-B_N2 back_30.0° C.) was obtained duringVT-XRPD test for Form IV sample (803759-02-B). The XRPD pattern isdisplayed in FIG. 11. After exposure to ambient condition, Form VIconverted to Form IV quickly (FIG. 9F). Since Form VI was unstable underambient condition, no further characterization data was collected forForm VI, and Form VI was speculated to be anhydrate.

Example 7 Preparation of Amorphous Form of Tegavivint

Amorphous sample (803759-04-B3 dry) was prepared by reverse anti-solventaddition in DMSO/H₂O system and vacuum drying at RT. The XRPD pattern isdisplayed in FIG. 12A. The mDSC result displayed in FIG. 12B showed theTg of the amorphous sample was 65.0° C. (middle temperature).

Example 8 Slurry Competition Experiments

To determine the most stable form under high water activity at RT,slurry competition of Form I, III, IV and V was performed in threesolvent systems (water, ACN/H₂O (1:1, v/v) and ACN/H₂O (1:3, v/v)) withhigh water activity at RT.

The detailed procedure was as follows:

1) Prepare saturated solution with Form I sample (803759-01-A) in threesolvent systems.

2) Add about 10 mg of each form into 1 mL corresponding saturatedsolution.

3) Slurry and check the XRPD of the wet cake after one day and six days.

The results are displayed in Table 2, FIG. 13A, FIG. 13B and FIG. 13C.The results showed:

1) Form IV was obtained from water system.

2) A new form was obtained from ACN/H₂O (1:1, v/v) system, which wasspeculated to be ACN solvate.

3) Form I was obtained from ACN/H₂O (1:3, v/v) system.

TABLE 2 Summary of slurry competition ACN/H₂O ACN/H₂O Starting MaterialH₂O (1:1, v/v) (1:3, v/v) Form I (803759-01- Form IV New form Form I A),Form III (speculated (803759-05-A1), to be ACN Form IV (803759-02-solvate) B) and Form V (803759-03-A 22Apr)

Example 9 Ball Milling Experiments

Previously, Form I was milled to a very small particle size and startedconverting to Form IV in aqueous suspension with unacceptable particlesize growth during storage. Therefore, ball milling of Form I and FormIV was performed to evaluate the form stability and particle sizegrowth. The detailed procedure of ball milling was as follows.

1. Suspend Form I and Form IV sample in 1% Poloxamer 188 water solution(50 mg/mL), separately.

2. Add ˜12 mL suspension into a 50-mL tube which contained milling beads(the volume of milling beads is around 30 mL). After 12 mL suspensionwas added, the liquid surface just covered the beads.

3. Roll the 50-mL tube (containing beads and suspension) at 5° C., RTand 60° C. with 30 rpm.

4. Sample ˜0.8 mL suspension at 1, 2, 4, 24 h using 1-mL syringe.

5. Test XRPD, PSD and PLM for the suspension.

The results displayed in Table 3 and in FIGS. 14A to 14T showed that:

1) form conversion was observed for Form IV at RT after 24 h and 60° C.after 2 h.

2) the particle size was decreased during milling.

3) Aggregation was observed for Form I at 5° C. and Form IV at 5° C. andRT after 24 h.

Therefore, ball milling of Form I sample at elevated temperature (60°C.) was recommended to reduce the particle size.

Additionally, the anticipated outcome is that milling of Form I at 60°C. or higher should prevent formation crystal seeds for the undesirableForm IV and result in a highly crystalline milled material that isannealed and free of high energy particles and free of amorphousmaterial.

Ball milling of Form IV at elevated temperature (60° C.) confirmedconversion to Form I.

TABLE 3 Summary of ball milling for Form I and Form IV at differenttemperatures Experiment ID 803759-15-A 803759-14-A1 803759-14-A2 InitialForm Form 1 Temperature, ° C. 5 RT 60 1 h Form Form I Form I Form I D 90(μm) 9.4 22.4 22.6 2 h Form Form I Form I Form I D 90 (μm) 9.0 21.4 6.64 h Form Form I Form I Form I D 90 (μm) 5.2 14.2 5.3 24 h Form Form IForm I Form I D 90 (μm) 5.6 2.8 2.0 Experiment ID 803759-15-B803759-14-B1 803759-14-B2 Initial Form Form IV Temperature, ° C. 5 RT 601 h Form Form IV Form IV Form IV D 90 (μm) 33.7 6.6 3.9 2 h Form Form IVForm IV Form I + IV D 90 (μm) 17.4 4.0 4.0 4 h Form Form IV Form IV FormI D 90 (μm) 5.8 3.4 2.0 24 h Form Form IV Amorphous Form I D 90 (μm) 9.85.8 0.6

To summarize, five crystal forms and amorphous sample of tegavivint wereobtained. The summary is displayed in Table 4.

TABLE 4 Summary of tegavivint polymorphs Endotherm Polymorph (ID) WeightLoss (%) (° C., onset) Form Form I 0.4 (150) ND Anhydrate (803759-01-A)Form III 8.2 (100) 64.5, 158.6* Unidentified (803759-05-A1) Form IV 8.4(100) 115.9^(#), 147.1* Hydrate (803759-13-B) Form V NA NA Unidentified(803759-03-A 22 Apr) Form VI (803759-02- NA NA Anhydrate B_N2back_30.0°C.) Amorphous NA 65.0** Amorphous (803759-04-B3 dry) ND: no thermalevent was observed before decomposition. *exothermic peak. ^(#)peaktemperature. **glass transition temperature (middle temperature). NA:the data was not collected

Appendix

Instruments and Methods

XRPD

For XRPD analysis, PANalytical X'Pert³ X-ray powder diffractometer wasused. The XRPD parameters used are listed in Table 5.

TABLE 5 Parameters for XRPD test Parameters X′ Pert# (reflection mode)X-Ray Cu, Kα, Kαl (Å): 1.540598: Kα2 (Å): 1.544426 Kα2/Kα1 intensityration: 0.50 X-Ray tube setting 45 KV, 40 mA Divergence slit 1/8° Scanmode Continuous Scan range (°2Theta) 3°~40° Scan step time (s) 46.667Step size (°2Theta) 0.0263° Test time (min) ~5 min

TGA, DSC and mDSC

TGA data were collected using a TA Discovery 5500/Q5000 TGA from TAInstruments. DSC and mDSC were performed using a TA Discovery 2500 DSCfrom TA Instruments. Detailed parameters used are listed in Table 6 andTable 7.

TABLE 6 Parameters for TGA and DSC test Parameters TGA DSC Method RampRamp Sample pan Aluminum, open Aluminum crimped Temperature RT~350° C.25~300° C. Heating rate 10° C./min 10° C./min Purge gas N₂ N₂

TABLE 7 Parameters for mDSC test Parameters mDSC Method ConventionalSample pan Aluminum crimped Temperature 25~200° C. Period 60 s Heatingrate 3° C./min Purge gas N₂

DVS

DVS was measured via a SMS (Surface Measurement Systems) DVS Intrinsic.The relative humidity at 25° C. were calibrated against deliquescencepoint of LiCl, Mg(NO₃)₂ and KCl. Parameters for DVS test were listed inTable 8.

TABLE 8 Parameters for DVS test Parameters DVS Temperature 25° C. Samplesize 10~20 mg Gas and flow rate N₂, 200 mL/min dm/dr 0.002%/min Min.dm/dt stability duration 10 min Max. equilibrium time 180 min RH range0% RH to 95% RH RH step size 10% RH from 0% RH to 95% RH 5% RH from 95%RH to 0% RH

PLM

PLM images were captured using Axio Scope A1 microscope from Carl ZeissGerman.

PSD

Microtrac S3500 with SDC (Sample Delivery Controller) was used for PSDtest and the method is shown in Table 9.

TABLE 9 PSD Method Parameters/Values Parameters/Values Distriction:Volume Run time: 10 sections/run Dispersive solvent: water Particle sizecoordinate: Standard Run number: 3 runs, average Solvent refractiveindex: 1.33 Transparency: Trans Residuals: Enabled Particle refractiveindex: 1.59 Flow rate: 60%* Particle Shape: Irregular Filter: OnSonication power: NA Sonication time: NA *60% of the maximum flow rate(65 mL/s)

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A crystalline form of a compound having the following formula:

wherein the crystalline form is designated as Form IV and has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 2θ angle values independently selected from the group consisting of 5.0+−0.2°; 7.5+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°.
 2. The crystalline form according to claim 1, wherein Form IV has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 2θ angle values independently selected from the group consisting of 5.0+−0.2°; 7.5+−0.2°; 7.7+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°.
 3. The crystalline form according to claim 2, wherein Form IV has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 2θ angle values independently selected from the group consisting of 5.0+−0.2°; 7.5+−0.2°; 7.7+−0.2°; 10.2+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°.
 4. The crystalline form according to claim 2, wherein Form IV has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 2θ angle values independently selected from the group consisting of 5.0+−0.2°; 7.5+−0.2°; 7.7+−0.2°; 10.2+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 18.0+−0.2°; 20.0+−0.2°; and 22.2+−0.2°.
 5. The crystalline form according to claim 2, wherein Form IV has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 2θ angle values independently selected from the group consisting of 5.0+−0.2°; 7.5+−0.2°; 7.7+−0.2°; 10.2+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 18.0+−0.2°; 20.0+−0.2°; 20.5+−0.2°; and 22.2+−0.2°.
 6. The crystalline form according to claim 1, wherein Form IV is characterized by having an endotherm with a peak maximum at approximately 115.9° C. by differential scanning calorimetry (DSC).
 7. The crystalline form according to claim 1, wherein Form IV is characterized by having an onset of exothermic peak at approximately 147.1° C. by differential scanning calorimetry (DSC).
 8. A nanosuspension of tegavivint prepared by a process comprising using Form IV as the starting material and milling Form IV at a temperature of between about 40° C. and about 60° C., wherein Form IV has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 2θ angle values independently selected from the group consisting of 5.0+−0.2°; 7.5+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°.
 9. A pharmaceutical composition, comprising a therapeutically effective amount of a crystalline form of tegavivint prepared by a process utilizing Form IV as the starting material, and a pharmaceutically acceptable excipient and/or diluent, wherein Form IV has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 2θ angle values independently selected from the group consisting of 5.0+−0.2°; 7.5+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°.
 10. A method for preventing, treating or ameliorating cancer or tumor metastasis in a mammal in need thereof comprising administering to said mammal an effective amount of the nanosuspension according to claim
 8. 11. The method of claim 10, wherein the cancer is acute myeloid leukemia (AML).
 12. A method for preventing, treating or ameliorating cancer or tumor metastasis in a mammal in need thereof comprising administering to said mammal an effective amount of the pharmaceutical composition according to claim
 9. 13. The nanosuspension of claim 8, wherein the Form IV is milled at a temperature of about 60° C.
 14. A nanosuspension of tegavivint prepared by a process comprising using Form IV as the starting material and milling Form IV at a temperature of between about 40° C. and about 60° C., wherein Form IV has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 2θ 8 angle values independently selected from the group consisting of 5.0+−0.2°; 7.5+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°, wherein the stable nanosuspension consists of Form I of tegavivint.
 15. A pharmaceutical composition, comprising a therapeutically effective amount of a crystalline form of tegavivint prepared by a process utilizing Form IV as the starting material, and a pharmaceutically acceptable excipient and/or diluent, wherein Form IV has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 2θ angle values independently selected from the group consisting of 5.0+−0.2°; 7.5+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°, wherein the crystalline form of tegavivint consists of Form I of tegavivint. 